Boosting the Potential of Chemotherapy in Advanced Breast Cancer Lung Metastasis via Micro‐Combinatorial Hydrogel Particles

Abstract Breast cancer cell colonization of the lungs is associated with a dismal prognosis as the distributed nature of the disease and poor permeability of the metastatic foci challenge the therapeutic efficacy of small molecules, antibodies, and nanomedicines. Taking advantage of the unique physiology of the pulmonary circulation, here, micro‐combinatorial hydrogel particles (µCGP) are realized via soft lithographic techniques to enhance the specific delivery of a cocktail of cytotoxic nanoparticles to metastatic foci. By cross‐linking short poly(ethylene glycol) (PEG) chains with erodible linkers within a shape‐defining template, a deformable and biodegradable polymeric skeleton is realized and loaded with a variety of therapeutic and imaging agents, including docetaxel‐nanoparticles. In a model of advanced breast cancer lung metastasis, µCGP amplified the colocalization of docetaxel‐nanoparticles with pulmonary metastatic foci, prolonged the retention of chemotoxic molecules at the diseased site, suppressed lesion growth, and boosted survival beyond 20 weeks post nodule engraftment. The flexible design and modular architecture of µCGP would allow the efficient deployment of complex combination therapies in other vascular districts too, possibly addressing metastatic diseases of different origins.

drug molecules. Loading is defined as the ratio between the mass of the loaded drug and the total mass of the nanoparticles. The encapsulation efficiency is defined as the percentage ratio between the mass of the loaded drug and the input drug.
For drug release studies, DTXL absorbance at 230 nm was considered. Specifically, SPN were dispersed in PBS and placed in dialysis cups (Slide-A-Lyzer MINI dialysis microtubes with a molecular cutoff of 10 kDa, Thermo Scientific) that were then exposed to PBS (4L), as the receptive phase. At different time points, 3 cups were removed from the solution, and their content was centrifuged to collect the SPN. These were then dissolved in an acetonitrile:water (1:1) solution before HPLC analysis. Release data are shown in the form of percentage cumulative release, which was obtained by adding the DTXL amount measured at a specific time point to all the previous time points and normalizing the resulting values for the initial amount of DTXL loaded into SPN.
NMR analysis for PEGDA-DTT conjugation. 5 mg of the PEGDA, DTT and PEGDA-DTT samples were dissolved in 0.5 ml of D2O and transferred into 5 mm SampleJet disposable tubes (Bruker). NMR experiments were performed at 298 K on a Bruker Avance III 600 MHz spectrometer equipped with 5 mm QCI cryoprobe with z shielded pulsed-field gradient coil, and operating at 600.13 MHz. 1H NMR spectra were acquired, after having applied a 30 degree of flip angle, with 16 transients, 65,536 points of digitalization, 1s of inter-pulses delay over a spectral width of 20.03 ppm (offset positioned at 6.18 ppm). related to the free thiol concentration via a standard curve. As depicted in Figure S2, the reaction of PEGDA with DTT was rapid and efficient. When DTT was introduced into buffered solutions containing PEGDA and allowed to react for 60 min, the thiol concentration rapidly dropped to 40% already after 1h. This residual concentration was then observed to stay constant for up to 24h, suggesting that not all the DTT was reacted with the PEGDA chains.
Consequently, free acrylate groups not reacted with DTT would be available in the polymeric matrix of the µCGP. The larger the PEGDA concentration, the larger the stiffness of the µCGP. Figure S4A shows how the increased polymer concentration leads to a more electron dense particle with a coloration varying from light gray to dark gray and black in the TEM images. The actual mechanical stiffness of the µCGP was quantified via AFM analyses. Figure S4B diluted into 20 mL of isotone and characterized at the Multisizer to count the number of µCGP per mL and assess their geometrical features. Figure S5A shows representative Multisizer profiles at 0 and 24h for all the tested conditions. Incubation in esterase determined a prompt degradation of µCGP with a 90% loss in particles already at 24h. The rapid enzymatic degradation of µCGP is related to the presence of the acrylate ester bonds proximal to the thioether hydrolytic bridges. µCGP degradation in PBS was slower with a loss of about 50% of the particles at 24h. Under this condition, µCGP degrade for the progressive hydrolysis of the dithiol 'bridges', which are hydrolytically labile. The degradation in PBS continued overtime reaching a loss in particle of 70% at 168h post incubation. In DI water, the degradation rate was even slower returning only a 40% loss in particles at 24h. The same experiments were also conducted for µCGP consisting of PEGDA only (no DTT) ( Figure S5B). Indeed, the particle degradation is much slower than for the PEG-DTT µCGP supporting the notion that the addition of the thiol bridges is fundamental for obtaining biodegradable microhydrogels.
Degradation data were further confirmed by confocal ( Figure S6) and SEM imaging ( Figure   S7). In particular, the confocal analysis shows how in water, the number of particles and the fluorescent intensity of µCGP was not significantly altered at 24h post incubation. On the other hand, in PBS, a 50% decrease in the number of particles was appreciated, while, in esterase, a massive drop in fluorescence and number of particles was observed after 24h, when it is evident that particles lose their native discoidal shape. Even more informative are the pictures taken at higher magnification in Figure S6B that show the progressive degradation of µCGP in an esterase solution: the particle morphology is completely lost at 72h.

Drug loading and release studies.
To boost the amount of drug per single µCGP, first the loading of DTXL in the spherical polymeric nanoconstructs was optimized. In particular, different input amounts of DTXL were considered to prepare the DTXL-SPN. As shown in Figure S8A, the increase in DTXL input did not affect the SPN physico-chemical properties returning a uniform and monodispersed size distribution at the DLS with an average size around 160 nm for all the different configurations. Conversely, the different input amounts of DTXL did affect the drug loading and the 3 mg input conditions were selected for the highest loading (600 µg). Lower and higher input amounts returned lower DTXL amounts ( Figure S8B). The encapsulation efficiencies for the 2 and 3 mg inputs were similar, and around 25% ( Figure   S8C). Interestingly, DTXL-µCGP obtained via the 3 mg DTXL-SPN (600 µg) led to a 2.5-fold increase in total drug loading as compared to DTXL-µCGP obtained via 2 mg DTXL-SPN (400 µg). The drug loading for the DTXL-µCGP was about five times higher than that typically measured for discoidal polymeric nanoconstructs (DTXL-DPN) previously realized by the authors using PLGA and molecular DTXL ( Figure S8D). 2 The release of the DTXL-SPN from the µCGP was also studied at physiological pH and mild acidic conditions and the results are presented in Figure S8E-F and directly compared to the release profiles obtained from SPN. 2 The results confirmed that the SPN confinement in the PEG matrix of µCGP reduces significantly the DTXL release, especially within the early time points and that an acidic environment accelerate the drug release overtime.          Importantly, these data demonstrate that the SPN biodistribution can be redirected upon their encapsulation within the hydrogel network of the CGP.
Third, UPCL-MS/MS was used to quantify the amount of DTXL accumulating within the lungs and the major organs. The results are presented in Figure 6 (main text) and expressed as the absolute percentage of the injected dose (%ID) and its value normalized by the mass of the tissue (%ID/g). In accordance with the biodistribution imaging studies and the in vivo therapeutic response, the amount of DTXL deposited within the lungs following a single DTXL-CGP injection was about two times higher than that measured for free-DTXL and DTXL-SPN. As per the other organs, in general, a low amount of DTXL was recovered in the case of free-DTXL and DTXL-SPN. This could be explained by the rapid DTXL clearance mostly through the hepatobiliary circulation. Conversely, the total amount of DTXL recovered in mice injected with DTXL-CGP was about 50% at the same time point, demonstrating the ability of this drug delivery platform to slowly release and preserve DTXL over time. Figure   S20 shows the direct comparison for all the analyzed organs and the different experimental groups with statistical analysis. respectively. Data are presented as the average ± SD, n ≥4 mice per experimental group.
Finally, for the fourth method, the preferential CGP accumulation in the lungs was observed via nuclear imaging. Figure S21 documents positron emission tomography (PET) images of CGP radiolabeled with 64 Cu accumulating within the TNBC-derived lung metastasis model. 64 Cu-CGP were realized by including PEG-DOTA conjugates for the chelation of Cu 2+radioactive ions with the DOTA cage. 64 Cu-CGP (4.7 MBq) were administered in mice bearing lung metastasis and scanned at 1 and 24h post injection (p.i.). Figure S21A shows